Evidence for the intracellular location of chloride channel (ClC)-type proteins: co-localization of ClC-6a and ClC-6c with the sarco/endoplasmic-reticulum Ca2+ pump SERCA2b.
Chloride channel protein (ClC)-6a and ClC-6c, a kidney-specific splice variant with a truncated C-terminus, are proteins that belong structurally to the family of voltage-dependent chloride channels. Attempts to characterize functionally ClC-6a or ClC-6c in Xenopus oocytes have so far been negative. Similarly, expression of both ClC-6 isoforms in mammalian cells failed to provide functional information. One possible explanation of these negative results is that ClC-6 is an intracellular chloride channel rather than being located in the plasma membrane. We therefore studied the subcellular location of ClC-6 isoforms by transiently transfecting COS and CHO cells with epitope-tagged versions of ClC-6a and ClC-6c. Confocal imaging of transfected cells revealed for both ClC-6 isoforms an intracellular distribution pattern that clearly differed from the peripheral location of CD2, a plasma-membrane glycoprotein. Furthermore, dual-labelling experiments of COS cells co-transfected with ClC-6a or -6c and the sarco/endoplasmic-reticulum Ca2+ pump (SERCA2b) indicated that the ClC-6 isoforms co-localized with the SERCA2b Ca2+ pump. Thus ClC-6a and ClC-6c are intracellular membrane proteins, most likely residing in the endoplasmic reticulum. In view of their structural similarity to proven chloride channels, ClC-6 isoforms are molecular candidates for intracellular chloride channels.
Buyse
G
G
Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium.
Trouet
D
D
Voets
T
T
Missiaen
L
L
Droogmans
G
G
Nilius
B
B
Eggermont
J
J
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D018118:Chloride Channels / Q000378:metabolism*; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D019084:Fluorescent Antibody Technique, Indirect; D018613:Microscopy, Confocal; D009865:Oocytes / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014162:Transfection; D014981:Xenopus
D018118:Chloride Channels; D000252:Calcium-Transporting ATPases